Site specific binding system, imaging compositions and methods

ABSTRACT

A method for ligand-based binding of lipid encapsulated particles to molecular epitopes on a surface in vivo or in vitro comprises sequentially administering (a) a site-specific ligand activated with a biotin activating agent; (b) an avidin activating agent; and (c) lipid encapsulated particles activated with a biotin activating agent, whereby the ligand is conjugated to the particles through an avidin-biotin interaction and the resulting conjugate is bound to the molecular epitopes on such surface. The conjugate is effective for imaging by x-ray, ultrasound, magnetic resonance or positron emission tomography. Compositions for use in ultrasonic imaging of natural or synthetic surfaces and for enhancing the acoustic reflectivity thereof are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/189,118, filed Nov. 9, 1998 which is a continuation of applicationSer. No. 08/854,308, filed May 12, 1997 now abandoned which is adivisional application of Ser. No. 08/488,743 filed Jun. 8, 1995, nowU.S. Pat. No. 5,690,907 dated Nov. 25, 1997.

BACKGROUND OF THE INVENTION

This invention relates to a novel site specific binding system and novelcompositions, and more particularly, to such a system and compositionswhich are useful in improved methods for ultrasonic imaging, drug orchemotherapeutic agent delivery, and diagnostic assays and detectionsystems.

Heretofore, with respect to ultrasonic imaging, although ultrasoniccontrast agents based upon “bubble” technology have been demonstrated todevelop an acoustic impedance mismatch by virtue of gas encapsulatedeither in protein (Feinstein et al., J. Am. Coll. Cardiol. 1990;16:316-324 and Keller et al., J. Am. Soc. Echo. 1989; 2:48-52),polysaccharide (Corday et al., J. Am. Coll. Cardiol. 1984; 3:978-85)biodegradable polymers (Schneider et al., Invest. Radiol., 1993;27:134-139 and Bichon et al., European Patent Application No.890810367.4: 1990) or lipids (D'Arrigo et al., J. Neurormag., 1991;1:134-139; Simon et al., Invest. Radiol., 1992; 27:29-34; and Unger etal., Radiology 1992; 195:453-456), no experimental evidence ofsite-specific targeting of an acoustic contrast or imaging agent withresultant changes in the acoustic properties of the targeted tissue,surface or support are known. This lack of results has occurred despitenumerous methods described in the literature for modifying such agentsfor targeting purposes, and the failure of past targeting approaches maybe due to the chemical nature of the agents, production processlimitations or particle instabilities.

Nongaseous acoustic contrast agents have been described including lipidemulsions (Fink et al., Ultrason. Imaging, 1985 7:191-197) liposomes(Lanza et al., J. Am. Coll. Cardiol., 1992 (abstract); 19 (3 Suppl A)114A), and perfluorocarbon emulsions (Mattrey et al., Radiology 1982;145: 759-762 and Mattrey et al., Ultrasound Med. 1983; 2:173-176). Aswith the contrast agents discussed above, no demonstration of sitetargeted emulsion or liposome has been reported. Again, such failure mayreflect instability of the particles, process incompatibilities or thechemical nature of the contrast agent. Lipid emulsions were evaluated byFink et al. supia and did not exhibit adequate echogenicity in studiesexamining hepatic imaging. A unique chemical formulation of liposomesdescribed by Lanza et al. supra was suggested to have the potential tobe a targetable ultrasonic contrast but such has not been demonstratedto date. Perfluorocarbon emulsions, Perflubron (perfluorooctylbromide,P100) and Flusol (perfluorodecalin and perfluorotripropylamine, F20)have been used as ultrasonic contrast agents and have been reported toaccumulate in liver, spleen and tumors secondary to phagocytic uptake ofemulsion particles at these sites (Mattrey et al. 1983, supra). Theseperfluorocarbon emulsions have also been noted to enhance Dopplersignals and opacify lumens. Fluorocarbons and fluorocarbon emulsions foruse as contrast agents are disclosed in U.S. Pat. Nos. 4,927,623,5,077,036, 4,838,274, 5,068,098, 5,114,703, 5,362,477, 5,362,478,5,171,755, 5,304,325, 5,350,571 and 5,403,575. However, no demonstrationof perfluorocarbon emulsions as a ligand targeted acoustic contrastsystem has been reported.

Previous descriptions of tissue or organ targeting in biomedicalultrasonics has referred to the collection of acoustically reflectiveparticles within or around structural tissue abnormalities. Localizedacoustic enhancement of tissue pathologies (e.g. malignancies) has notbeen ligand-directed but rather has depended upon differential dynamicrates of particle uptake and/or clearance between normal and malignanttissues. Such contrast agents have included aqueous solutions (Ophir etal., Ultrason. Imaging 1979, 1:265-279; Ophir et al., Ultrasound Med.Biol. 1989, 15:319-333; and Tyler et al., Ultrason. Imaging, 3:323-329),emulsions (Fink et al. Ultrason. Imaging, 1985, 7:191-197), andsuspensions (Mattrey et al. 1982 supra and Mattrey et al., Radiology,1987, 163:339-343). Although the possibility of ligand-directedultrasonic contrast targeting with acoustically reflective liposomes hasbeen suggested, no successful applications of this concept have beenreported (Lanza et al. 1992, supra and Valentini et al., J. Am. Coll.Cardiol., 1995, 25:16A). Previous approaches to targeting in vivo ofparticles have involved direct conjugation of a ligand (e.g. monoclonalantibody) to a vesicle by a variety of methods (see, for example,Torchlin et al., Biochem. Biophys. Res. Commun. 1978, 85:983-990; Endohet al., J. Immunol. Methods, 1981, 44:79-85; Hashimoto et al., J.Immunol. Methods, 1983, 62:155-162 and Martin et al., Biochemistry,1981, 20:4229-4238).

There remains a need for new and improved methodologies for ligand-basedbinding systems which can be adapted as an ultrasonic contrast systempermitting detection of molecular moieties such as peptides,carbohydrates or nucleic acids and whose uses can range fromultrasound-based ELISA-like laboratory diagnostic assays in liquid andsolid phase systems and in cell cultures; electrophoretic,chromatographic and hybridization detection systems to the detection ofthrombi, infections, cancers and infarctions in patients with the use ofconventional ultrasonic imaging methods.

SUMMARY OF THE INVENTION

Among the several objects of the invention may be noted the provision ofa novel method for ligand-based binding of lipid encapsulated particlesto molecular epitopes on a surface in vivo or in vitro, the provision ofsuch a method in which the ligand is conjugated to the lipidencapsulated particles through an avidin-biotin interaction and theresulting conjugate is bound to molecular epitopes on a surface; theprovision of such a method which is useful for enhancing the acousticreflectivity of a biological surface for ultrasonic imaging; theprovision of a method of this type wherein the conjugate formed iseffective for imaging by x-ray, ultrasound, magnetic resonance orpositron emission tomography; the provision of compositions for use inultrasonic imaging of a biological surface and for enhancing theacoustic reflectivity of such a surface; the provision of ultrasoniccontrast agents which become highly reflective when bound to the desiredsite or biological surface through the ligand-based binding system ofthe invention; and the provision of such methods and compositions whichare capable of targeting and altering the echogenic properties of atissue surface for improved and specific identification of pathologicalprocesses. Other objects will be in part apparent and in part pointedout hereinafter.

Briefly, in its broadest embodiment, the present invention is directedto a method for ligand-based binding of lipid encapsulated particles tomolecular epitopes on a surface in vivo or in vitro which comprisessequentially administering (a) a site-specific ligand activated with abiotin activating agent; (b) an avidin activating agent; and (c) lipidencapsulated particles activated with a biotin activating agent, wherebythe ligand is conjugated to the particles through an avidin-biotininteraction and the resulting conjugate is bound to the molecularepitopes on such surface. The conjugate is effective for imaging byx-ray, ultrasound, magnetic resonance or positron emission tomography.In a more specific embodiment, the invention is directed to a method forenhancing the acoustic reflectivity of a biological surface through thesequential administration of the above-noted components whereby theresulting conjugate is bound to a natural or synthetic surface toenhance the acoustic reflectivity thereof for ultrasonic imaging. Theinvention is also directed to compositions for use in ultrasonic imagingof such surfaces and for enhancing the acoustic reflectivity thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing changes in aggregate particle size ofbiotinylated and control perfluorocarbon emulsions with increasingavidin concentration;

FIG. 2 shows ultrasonic images of control and biotinylatedperfluorocarbon emulsion before and after the addition of avidin;

FIG. 3 is a graphic illustration of dialysis tubing images and region ofinterest placement for gray scale analysis;

FIG. 4 is a graph showing changes in average pixel gray scale associatedwith the addition of avidin to control or biotinylated perfluorocarbonemulsion;

FIG. 5 is a graph showing the effect of control and biotinylatedperfluorocarbon emulsion on apparent backscatter transfer function andintegrated backscatter of avidinized nitrocellulose membranes;

FIG. 6 is a graph showing the apparent backscatter transfer function ofbiotinylated and control perfluorocarbon emulsions targeted to D-dimercovalently conjugated to nitrocellulose membranes;

FIG. 7 is a graph showing the apparent backscatter transfer function(dB) of biotinylated and control perfluorocarbon emulsions at lowultrasonic frequencies;

FIG. 8 is a graph showing the apparent backscatter transfer function ofbiotinylated and control perfluorocarbon large particle size emulsionstargeted to avidinized nitrocellulose membranes;

FIG. 9 shows ultrasonic images of plasma thrombi before and afterexposure to control or biotinylated emulsions;

FIG. 10 is a graph showing the average pixel grayscale level of plasmathrombi pretargeted with antifibrin monoclonal antibody and exposed tocontrol or biotinylated perfluorocarbon emulsion;

FIG. 11 shows ultrasonic images of femoral artery thrombus acousticallyenhanced with biotinylated perfluorocarbon emulsion in vivo;

FIG. 12 is a graph showing the net change in apparent backscattertransfer function of biotinylated and control perfluorocarbon emulsionstargeted to prostate specific antigen in prostatic carcinoma relative tonormal regions;

FIG. 13 is a graph showing the net change in integrated backscatterbetween normal prostatic stroma and cancer regions for control versusbiotinylated perfluorocarbon emulsions;

FIG. 14 is a graph showing the net change in apparent backscattertransfer function of biotinylated and control perfluorocarbon emulsionstargeted to OC-125 antigen in ovarian carcinoma relative to normalregions;

FIG. 15 is a graph showing the net change in integrated backscatterbetween normal ovarian tissue and carcinoma regions for control versusbiotinylated perfluorocarbon emulsions;

FIG. 16 shows ultrasonic and optical images of tonsil usingperfluorocarbon contrast and horseradish peroxidase targeted toepithelium with anticytokeratin antibodies; and

FIG. 17 shows peak detected ultrasonic images of tonsil epitheliumacoustically enhanced with anticytokeratin antibody targetedperfluorocarbon emulsion.

FIG. 18 is a graph showing backscattered power from plasma clots afterthe one step fibrin targeted emulsion, the backscatter being computedfrom the average of the frequency-dependent backscatter transferfunction over the useful bandwidth of the transducer;

FIG. 19 shows scanning electron microscope images of cell culturestreated with no (FIG. 19A) or untargeted control emulsion (FIG. 19B),tissue factor targeted specifically bound to the aortic smooth musclecells (FIG. 19C) and binding specifically blocked by anti-tissue factorantiserum (FIG. 19D);

FIG. 20 shows ultrasonic images of vessels before and after treatmentwith targeted conjugated emulsion; and

FIG. 21 shows the results of staining which revealed a pattern oflocalization of tissue factor in balloon-injured vessels in the media(arrows) at the site of acoustic contrast enhancement, as well as on theinternal surface associated with inflammatory cells (FIG. 21A; L-lumen)and control uninjured vessels exhibiting staining primarily on theadventitia and not in the tunica media (FIG. 21B).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, it has now been found that aligand-based binding system having broad application may be achievedthrough ligand-based binding of lipid encapsulated particles tomolecular epitopes on a surface in vivo or in vitro by sequentiallyadministering (a) a site-specific ligand activated with a biotinactivating agent; (b) an avidin activating agent; and (c) lipidencapsulated particles activated with a biotin activating agent, wherebythe ligand is conjugated to the lipid encapsulated particles through anavidin-biotin interaction or complexing and the resulting conjugate isbound to the molecular epitopes on the surface. The ligand-based bindingsystem of the present invention thus permits detection of molecularmoieties such as peptides, carbohydrates or nucleic acids with aspecific ligand probe (e.g. an antibody or antibody fragment) complexedor conjugated with avidin and biotin, the latter being carried by lipidencapsulated particles (e.g. biotinylated lipid encapsulated emulsion orliposome). The ligand-based binding system of the invention may beemployed in an ultrasonic contrast agent system, ultrasound-basedELISA-like laboratory diagnostic assays in liquid and solid phasesystems and in cell cultures, electrophoretic, chromatographic andhybridization detection systems, and for the detection of thrombi,infections, cancers and infarctions in patients with the use ofconventional ultrasonic imaging methods. The invention may also beapplied for therapeutic purposes by delivery of chemotherapeutic agentsor drugs to desired sites due to the specificity of the binding systemcoupled with the ability to monitor the progress of the therapeutictreatment through repeated imaging at such sites. In this regard, theabove-referred to conjugate of the ligand to the lipid encapsulatedparticles through an avidin-biotin interaction or complexing iseffective for imaging by x-ray, ultrasound, magnetic resonance orpositron emission tomography.

In one embodiment of the invention, there is provided a method forenhancing the reflectivity of a biological surface by sequentiallyadministering to the surface (a) a site-specific ligand activated with abiotin activating agent; (b) an avidin activating agent; and (c) lipidencapsulated particles activated with a biotin activating agent; wherebythe ligand is conjugated to the lipid encapsulated particles through anavidin-biotin interaction and the resulting conjugate is bound to thebiological surface to enhance the acoustic reflectivity thereof forultrasonic imaging. This novel triphasic approach utilizes anavidin-biotin interaction to permit administration of the targetingligand separate from the acoustic lipid encapsulated particles. In aspecific application of the method in accordance with the invention, abiotinylated ligand is first systemically administered to a patient topretarget the tissue or biological surface of interest and to circulatefor a period of time necessary or sufficient to optimize the percentagebound. In the second phase, avidin is administered, circulates and bindsto the biotinylated ligand attached to the target tissue or surface andto any residual, free circulating ligand. Avidin cross-linking increasesthe avidity and stability of the ligand on the target tissue or surfacewhile promoting the rapid clearance of circulating avidin-ligandcomplexes via the reticuloendothelial system. In the third phase, thebiotinylated lipid encapsulated particles are administered, binding toavidin through unoccupied biotin binding sites, and imparting increasedacoustic contrast to the targeted tissue surface. Repeated sequentialadministration of avidin and the biotinylated lipid encapsulatedparticles may be carried out to amplify the acoustic contrast effect ofthe lipid encapsulated particles bound to the targeted surface.

In the practice of the invention, the ligand employed may be, forexample, constituted by monoclonal or polyclonal antibodies, viruses,chemotherapeutic agents, receptor agonists and antagonists, antibodyfragments, lectin, albumin, peptides, hormones, amino sugars, lipids,fatty acids, nucleic acids and cells prepared or isolated from naturalor synthetic sources. In short, any site-specific ligand for anymolecular epitope or receptor to be detected through the practice of theinvention may be utilized.

The ligand is activated with a biotin activating agent. As employedherein, the term “biotin activating agent” or “biotinylated” encompassesbiotin, biocytin and other biotin analogs such as biotin amido caproateN-hydroxysuccinimide ester, biotin 4-amidobenzoic acid, biotinamidecaproyl hydrazide and other biotin derivatives and conjugates. Otherderivatives include biotin-dextran,biotin-disulfide-N-hydroxysuccinimide ester, biotin-6 amido quinoline,biotin hydrazide, d-biotin-N hydroxysuccinimide ester, biotin maleimide,d-biotin p-nitrophenyl ester, biotinylated nucleotides and biotinylatedamino acids such as Nε-biotinyl-1-lysine.

In the second phase, as previously mentioned, an avidin activating agentis administered. As employed herein, the term “avidin activating agent”or “avidinized” encompasses avidin, streptavidin and other avidinanalogs such as streptavidin or avidin conjugates, highly purified andfractionated species of avidin or streptavidin, and non or partial aminoacid variants, recombinant or chemically synthesized avidin analogs withamino acid or chemical substitutions which still accommodate biotinbinding.

The lipid encapsulated particles or contrast agent employed in the thirdphase may be constituted, for example, by a biotinylated emulsion orliposome which may contain a gas, liquid or solid. In a specificexample, the lipid encapsulated particles may be constituted by aperfluorocarbon emulsion, the emulsion particles having incorporatedinto their outer coating a biotinylated lipid compatible moiety such asa derivatized natural or synthetic phospholipid, a fatty acid,cholesterol, lipolipid, sphingomyelin, tocopherol, glucolipid,stearylamine, cardiolipin, a lipid with ether or ester linked fattyacids or a polymerized lipid. Thus, the biotinylated contrast agentconstituting the lipid encapsulated particles may be produced byincorporating biotinylated phosphatidylethanolamine into the outer lipidmonolayer of a perfluorocarbon emulsion.

Perfluorocarbon emulsions are particularly well suited for biomedicalapplications and for use in the practice of the present invention. Theyare known to be stable, biologically inert and readily metabolized,primarily by trans-pulmonic alveolae evaporation. Further, their smallparticle size easily accommodate transpulmonic passage and theircirculatory half-life (4-8 hours) advantageously exceeds that of otheragents. Also, perfluorocarbons have been used to date in a wide varietyof biomedical applications, including use as artificial bloodsubstitutes. For use in the present invention, various fluorocarbonemulsions may be employed including those in which the fluorocarbon is afluorocarbon-hydrocarbon, a perfluoroalkylated ether, polyether or crownether. Useful perfluorocarbon emulsions are disclosed in U.S. Pat. Nos.4,927,623, 5,077,036, 5,114,703, 5,171,755, 5,304,325, 5,350,571,5,393,524, and 5,403,575 and include those in which the perfluorocarboncompound is perfluorotributylamine, perfluorodecalin,perfluorooctylbromide, perfluorodichlorooctane, perfluorodecane,perfluorotripropylamine, perfluorotrimethylcyclohexane or otherperfluorocarbon compounds. Further, mixtures of such perfluorocarboncompounds may be incorporated in the emulsions utilized in the practiceof the invention. As a specific example of a perfluorocarbon emulsionuseful in the invention may be mentioned a perfluorodichlorooctaneemulsion wherein the lipid coating thereof contains betweenapproximately 50 to 99.5 mole percent lecithin, preferably approximately55 to 70 to mole percent lecithin, 0 to 50 mole percent cholesterol,preferably approximately 25 to 45 mole percent cholesterol andapproximately 0.5 to 10 mole percent biotinylatedphosphatidylethanolamine, preferably approximately 1 to 5 mole percentbiotinylated phosphatidylethanolamine. Other phospholipids such asphosphatidylserine may be biotinylated, fatty acyl groups such asstearylamine may be conjugated to biotin, or cholesterol or other fatsoluble chemicals may be biotinylated and incorporated in the lipidcoating for the lipid encapsulated particles. The preparation of anexemplary biotinylated perfluorocarbon for use in the practice of theinvention is described hereinafter in accordance with known procedures.

When the lipid encapsulated particles are constituted by a liposomerather than an emulsion, such a liposome may be prepared as generallydescribed in the literature (see, for example, Kimelberg et al., CRCCrit. Rev. Toxicol. 6, 25 (1978) and Yatvin et al., Medical Physics,Vol. 9, No. 2, 149 (1982)). Liposomes are known to the art and generallycomprise lipid materials including lecithin and sterols, eggphosphatidyl choline, egg phosphatidic acid, cholesterol andalpha-tocopherol.

With respect to the particle size of the lipid encapsulated particlesconstituted by a perfluorocarbon emulsion or liposome, the particle sizemay range between approximately 0.05 to 5 microns and preferably betweenapproximately 0.05 and 0.5 micron. Small size particles are thuspreferred because they circulate longer and tend to be more stable thanlarger particles.

As indicated, the ligand is conjugated to the lipid encapsulatedparticles or perfluorocarbon emulsion through an avidin-biotininteraction. The ligand may also be conjugated to the emulsion directlyor indirectly through intervening chemical groups or conjugated directlyor indirectly to biotin or a biotin analog through intervening chemicalgroups such as an alkane spacer molecule or other hydrocarbon spacer.The use of spacer molecules between the ligand and biotin or betweenbiotin and the emulsion is not required but aids in rendering the biotinmore available for binding to avidin.

In accordance with the broadest aspect of the invention, it has beenfound that liquid perfluorocarbon emulsions have very poor intrinsicechogenicity when free in suspension, but when bound to a surface, theyincrease the acoustic reflectivity of the surface. By conjugating theligand directly to the emulsion and binding the resultingligand-emulsion conjugate to a surface, enhanced acoustic reflectivityis realized for ultrasonic imaging.

As previously mentioned, the emulsion or liposome constituting the lipidencapsulated particles or vesicles may contain a gas, liquid or solid.The gas may be nitrogen, oxygen, carbon dioxide or helium and may, forexample, be evolved from the fluorocarbon component of the emulsionsdescribed above.

Alternatively, but less preferably, the ligand-based binding method ofthe invention may be carried out by sequentially administering asite-specific ligand activated with a biotin or avidin activating agentand lipid encapsulated particles activated with a biotin or avidinactivating agent, a biotin activating agent being used where an avidinactivating agent was employed in the first step and an avidin activatingagent being used where a biotin activating agent was employed in thefirst step. The direct conjugation of the ligand to a perfluorocarbonemulsion, for example, is less preferable since it may accelerate invivo clearance of the emulsion contrast agent.

In the practice of the invention, it has been unexpectedly found thatthe individual components of the ultrasonic contrast agents as describedabove are poorly reflective or have low echogenicity in the bloodstreambut become highly reflective when the ligand-avidin-emulsion complex isformed in vivo at the desired site or biological surface and therebysubstantially enhances the acoustic reflectivity thereof for ultrasonicimaging. This is in sharp contrast to previously known sonographiccontrast agents which are inherently bright or of high reflectivity inthe bloodstream. The improved acoustic reflectivity achieved through thepresent invention provides the advantage of enhancing thesignal-to-noise ratio because the background contrast from lipidencapsulated particles in the blood is minimal. Thus, the presentinvention offers an improved noninvasive method for forming an acousticcontrast agent which can be targeted in vitro or in vivo and which whenbound to a specific desired site alters the acoustic reflectivity of atissue surface or support media in a manner detectable with ultrasonictransducers suitable for biomedical and diagnostic applications within afrequency range of at least 5 to 50 MHz (nominal center frequencies maybe wider ranging based on the knowledge that these are broad bandtransducers). The method of the invention advantageously provides apractical means for detecting any molecular epitope or receptor forwhich a biotinylated monoclonal antibody or other ligand is availablewithout the need for use of ionizing radiation with or withoutassociated invasive procedures in various clinical applications andwhile employing standard, commercially available ultrasonic technology.The present invention does not employ ultrasonic contrast systems oragents to delineate blood flow as in the prior art but rather to detectphysiologic and pathologic events by sensing the accumulation of thecontrast agent at specific binding sites.

In the application of the invention to diagnostic assays such asultrasound-based ELISA-type laboratory diagnostic assays in liquid andsolid phase systems, the surface on which ligand-based binding of lipidencapsulated particles to molecular epitopes occurs may be, for example,nylon, nitrocellulose membranes or a gel as well as a biologicalsurface.

The ligand-based binding system of the invention may also be applied toprovide a chemotherapeutic agent or gene therapy delivery systemcombined with ultrasonic imaging. For example, chemotherapeutic agentsor immune activating drugs such as tissue plasminogen activator,adriamycin, vincristine, urokinase, streptokinase, methotrexate,cytarabine, thioguanine, doxorubicin, 5-fluorouracil, cisplatin,etoposide, ifosfamide, asparginase, deoxycoformycin, hexamethyl melamineand including radioactive agents may be incorporated in the lipidencapsulated particles and become part of the conjugate bound to aspecific biological surface site for therapeutic action. The presentinvention would also advantageously permit the site to be ultrasonicallyimaged in order to monitor the progress of the therapy on the site andto make desired adjustments in the dosage of therapeutic agentsubsequently directed to the site. The invention thus provides anoninvasive means for the detection and therapeutic treatment ofthrombi, infections, cancers and infarctions in patients while employingconventional ultrasonic imaging systems.

The following examples illustrate the practice of the invention.

EXAMPLE 1

The procedure for preparing a biotinylated lipid encapsulatedperfluorodichlorooctane emulsion for use in ultrasound imaging is asfollows.

The biotinylated lipid perfluorodichlorooctane (PFDCO) emulsion iscomprised of the following components: PFDCO (40% v/v), safflower oil(2.0% w/v), a surfactant co-mixture (2.0% w/v) and glycerin (1.7% w/v).The surfactant co-mixture is composed of approximately 64 mole %lecithin, 35 mole % cholesterol and 1 mole %N-(6-biotinoyl)amino)hexanoyl)dipalmitoyl-L-alpha-phosatidylethanolamine.These components are weighed together into a test tube and dissolved inchloroform. The chloroform is stripped from the material and theresulting surfactant mixture is dried in a 50° C. vacuum oven overnight.The co-mixture is dispersed into water by sonication resulting in aliposome suspension. The suspension is transferred into a 30 mL capacityblender cup (Dynamics Corporation of America, New Hartford, Conn.) alongwith the PFDCO and oil. The mixture is blended for 30-60 seconds to apre-emulsion. The preemulsified sample is transferred to the reservoirof a microfluidizer, model S110 (Microfluidics, Newton, Mass.), andemulsified for three minutes at 10,000 psi. To prevent the emulsion fromheating excessively during homogenization, the shear valve and mixingcoil of the microfluidizer are immersed in a room temperature water bathduring processing. The final temperature of the emulsion isapproximately 35° C. The finished emulsion is bottled in 10 mL serumvials, blanketed with nitrogen gas and sealed with stopper/crimp seal.The average particle size of the finished product, measured by a laserlight scatter particle sizer (Brookhaven Instruments Corporation,Holtsville, N.Y.), is 250 nm.

EXAMPLE 2

The incorporation of biotinylated phosphatidylethanolamine into theencapsulating lipid monolayer of perfluorocarbon emulsion is prepared asdescribed in Example one and demonstrated to increase aggregate particlesize in the presence of titrated concentrations of avidin (Pierce,Rockford, Ill. 61105). An identically prepared control emulsion isprepared which incorporates nonbiotinylated phosphatidylethanolamineinto the outer lipid monolayer of the perfluorocarbon emulsion. Avidinis resuspended in isotonic phosphate buffered saline (PBS, Fisher Inc.,Fair Lawn, N.J.). Within a polystyrene cuvette, a 3.0 ml reactionmixture is prepared containing PBS, biotinylated or controlperfluorocarbon emulsion (20 μl) and avidin, at 0.0, 0.5, 1.0, 1.5 or2.0 μg/ml. Contents are mixed by gentle inversion and react for thirtyminutes at room temperature. Emulsion particle sizes are determined intriplicate with a Brookhaven BI-90 particle size analyzer (Holtsville,N.Y.) at 37° C. Aggregate particle size of the biotinylated emulsionincreased progressively from a baseline of 263±2.54 nm to greater than2000 nm with increasing concentration of avidin (FIG. 1). Markedflocculation and sedimentation are noted when avidin concentrationsexceed 2.0 μg/ml. The particle size of the control emulsion is 234±3.81nm in diameter and addition of 2.0 μg of avidin to the reaction mixturedoes not affect particle size. These results clearly demonstrate thatthe biotinylated phosphatidylethanolamine is incorporated and orientedappropriately into the outer lipid monolayer of the perfluorocarbonemulsion and that surface biotins are adequately available to avidin inthe media. Multiple biotin binding sites on the avidin molecule as wellas multiple biotin residues on the surface of the emulsion progressestowards a rapid complexing of particles in vitro.

EXAMPLE 3

Biotinylated perfluorocarbon emulsion particles, approximately 250 nm indiameter, with low independent acoustic reflectivity, are complexed withavidin in solution which eventuates aggregation and enhancesechogenicity. Biotinylated and control perfluorocarbon emulsion (200 μl)prepared as described previously are diluted in PBS (15 ml) and placedwithin dialysis tubing (Spectra/Por 4, 25 mm, MWCO 12,000-14,000,Spectrum Medical Industries, Inc., Los Angeles, Calif.), ultrasonicallyimaged within a PBS water bath at room temperature using a 7.5 MHzfocused transducer and a Hewlett Packard (HP) Sonos 2500 Phased ArrayImaging System (Andover, Mass.). Real-time images are recorded to SVHSvideo tape for subsequent image analysis. Pixel grayscale andhomogeneity are assessed on selected freeze-frame images using NIH Image1.47 (National Institutes of Health). Avidin (30 μg/ml) is added to eachemulsion suspension, mixed by gentle inversion and allowed to complexfor 30 minutes. The emulsion suspensions are optically opaque butultrasonically undetected prior to the addition of avidin. Complexing ofthe biotinylated perfluorocarbon emulsion ensues rapidly with theaddition of avidin and a white, flocculant precipitate soon appears.Avidin induces no changes in control emulsion suspension. Insonificationof the suspensions reveals that the biotinylated perfluorocarbonemulsion particles opacify the dialysis tubing; whereas, the controlparticles are not appreciated acoustically (FIG. 2). Gray scale echointensity analysis of freeze-frame images of the control andbiotinylated emulsion suspensions before and after avidin are summarizedin FIGS. 3 and 4. The increased average grayscale level of thebiotinylated emulsion (71.3±22.1) suspension relative to its pre-avidinpixel gray scale level (2.2±4.4) demonstrates the acoustic enhancementachieved. Average pixel gray scale levels of the control emulsion before(3±7.33) and after (1.0±1.3) avidin addition are similar. These resultsdemonstrate the low acoustic reflectivity of the perfluorocarbonemulsion when imaged as independent particles in comparison with theenhanced echogenic nature of the aggregated biotinylated particles. Thelack of acoustic change in the control emulsion suspension in thepresence of avidin confirms the ligand specificity of the biotinylatedemulsion.

EXAMPLE 4

Biotinylated perfluorocarbon emulsion, approximately 250 nm diameter,are specifically targeted to avidin, covalently bound to a modifiednitrocellulose membrane and increases the acoustic reflectivity of themembrane surface at high ultrasonic frequencies (30 to 60 MHz). Briefly,nitrocellulose membranes (S+S NC™, Schleicher & Schuell, Keane, N.H.)were conjugated to avidin using a diaminohexane (Sigma Chemical Co., St.Louis, Mo.) spacer and glutaraldehyde (Sigma Chemical Co., St. Louis,Mo.) activation as described by Masson et al. (Electrophoresis 1993, 14,860-865). Nitrocellulose discs (2 cm diameter) are soaked in 2.5%diaminohexane dissolved in deionized water for 60 minutes with constantslow rotary agitation. Membranes are washed with 1M acetic acid for 6-7hours followed by an 18+ hour deionized water wash with constantagitation. The membranes are placed in 1% glutaraldehyde in 0.1M sodiumbicarbonate buffer, pH 10.0 for 15 minutes then washed for three hourswith deionized water. Nitrocellulose membranes are stored and dried at4° C. until use; storage does not exceed three days.

Fifty (50) μl of avidin (250 μg) are spotted dropwise upon the center ofsix membranes with a microliter syringe and allowed to dry. Eachmembrane is extensively washed with a 0.1% Tween-20 (Sigma Chemical Co.,St. Louis, Mo.) in PBS then placed in 3% bovine serum albumin (BSA,crystallized, Sigma Chemical Company, St. Louis, Mo.) dissolved inPBS-0.1% Tween-20 for 20 minutes to blockade nonspecific protein bindingsites around the periphery of the disc. After the BSA blockade, eachdisc is extensively washed with PBS and placed in 300 μl of eitherbiotinylated or control perfluorocarbon emulsion suspended in 4 ml PBSfor 20 minutes. Unbound emulsion is removed in serial PBS washes. Eachdisc is reexposed to avidin and control or biotinylated emulsion toensure saturated coverage of the nitrocellulose surface. Thenitrocellulose discs are washed and stored in PBS at 4° C. until imagedwith acoustic microscopy.

For acoustic microscopic imaging, each nitrocellulose disc is placedflat above a polished stainless steel plate in a polystyrene holder witha 2×2 cm central window removed. The mounted specimen is immersed intoPBS at ambient temperature for ultrasonic insonification. A customdesigned acoustic microscope, utilizing a 50 MHz (nominal frequency)broadband, focused, piezoelectric delay-line transducer (¼ inchdiameter, ½ inch focal length, Model V390, Panametrics Co., Waltham,Mass.) operated in the pulse-echo mode is utilized for insonification.Backscattered radio frequency (RF) data is collected and digitized at500 megasamples per second utilizing a Tektronix DSA 601 digitizingoscilloscope (Beaverton, Oreg.) with 8-bit resolution. A variable gainsystem is used to increase the effective dynamic range of thisdigitizer. Radio frequency data are acquired from approximately 100independent sites from each region of interest with 100 micron lateralstep resolution.

A radio frequency peak-detected scan of the data is converted into agray scale (0=lowest scattering, 255=highest scattering) map to allowselection of regions of interest for integrated backscatter analysis.Radio frequency (RF) ultrasonic data are stored in a raster scan formatand analyzed with custom software. Segments of the RF lines are gatedfor integrated backscatter analysis to encompass the front and backsurfaces of the nitrocellulose disc. The data are multiplied by arectangular window and their power spectra are determined byfast-Fourier transformation. The power spectra from the specimensreferenced to the power spectrum returned from a near-perfect steelplanar reflector and the frequency-dependent backscatter transferfunction across the useful bandwidth of the transducer (30 to 60 MHz)are computed and expressed in decibels relative to acoustic scatteringfrom the near perfect steel plate reflector (Wong et al., Ultrasound inMed & Biol. 1993; 19: 365-374). Integrated backscatter (IB) is computedas the average of the frequency-dependent backscatter transfer functionacross the useful bandwidth of the transducer.

Discs incubated with biotinylated perfluorocarbon emulsion have centralregions with high acoustic scattering in comparison with the peripheral(i.e. background) regions of the same disc. Nitrocellulose discsincubated with the control emulsion have no central high scatteringregions and no differences in acoustic character is detected by changesin the RF signature between the central and peripheral regions of thedisc. IB from the centrally-located, biotinylated emulsion region(−17.8±0.2 db) is 6.3±0.1 dB (4-fold) greater (p<0.05) than IB from theanalogous region on the control disc (−24.1±0.2 dB). Thefrequency-dependent variation in apparent backscatter transfer function(mean±SEM) from the avidin spotted regions of the biotinylated andcontrol emulsion discs are presented in FIG. 5. A smooth andconsistently greater acoustic response is noted across the frequencyspectrum due to the bound biotinylated emulsion. These resultsdemonstrate the effectiveness of the biotinylated perfluorocarbonemulsion to specifically target a surface bound antigen and dramaticallyalter the acoustic reflectivity of the surface with the bathing medium,increasing the ultrasonic backscattered power at high frequencies.

EXAMPLE 5

Biotinylated perfluorocarbon emulsion (250 nm diameter) is specificallytargeted to D-dimer covalently attached to a modified nitrocellulosemembrane utilizing a biotinylated anti-D-dimer F_((ab)) fragment-avidincomplex and results in a marked increase in the acoustic power reflectedfrom the surface. D-dimer is covalently linked to nitrocellulose discsmodified with a diaminohexane spacer arm and activated withglutaraldehyde as previously described in Example 4. Fifty (50) μg ofD-dimer is spotted with a microliter syringe upon the center of three ofsix membranes and allowed to air dry. Unbound D-dimer is exhaustivelywashed from the membranes with phosphate buffered saline (PBS)-0.1%Tween-20. Nonspecific protein binding sites of all membranes are blockedwith 3% bovine serum albumin (BSA) in PBS-0.1% Tween-20 for 20 minutesfollowed by serial PBS washes. D-dimer spotted membranes are incubatedwith 12.5 μg biotinylated anti-D-dimer F_((ab)) antibody in 4.0 ml 3%BSA for 2 hours, washed with PBS buffer and then incubated with 250 μgavidin in 4 ml PBS for 30 min. After removing unbound avidin with PBSwashes, the discs are exposed to either biotinylated or controlperfluorocarbon emulsion (300 μl) in 4.0 ml PBS for 20 minutes. Excessemulsion is removed with PBS buffer washes. Discs are reexposed toavidin and perfluorocarbon emulsion as described above and the membranesare stored in PBS at 4° C. until imaging.

For acoustic microscopic imaging, each nitrocellulose disc is placedflat above a polished stainless steel plate in a polystyrene holder,immersed in PBS at ambient temperature, and insonified with a customdesigned acoustic microscope, utilizing a 50 MHz (nominal frequency)broadband, focused, piezoelectric delay-line transducer (¼ inchdiameter, ½ inch focal length, Model V390, Panametrics Co., Waltham,Mass.) operated in the pulse-echo mode. Backscattered radio frequency(RF) data is collected and digitized at 500 megasamples per secondutilizing a Tektronix DSA 601 digitizing oscilloscope (Beaverton, Oreg.)with 8-bit resolution. A variable gain system is used to increase theeffective dynamic range of this digitizer. Radio frequency data areacquired from approximately 100 independent sites from each region ofinterest with 100 micron lateral step resolution.

A radio frequency peak-detected scan of the data is converted into agray scale (0=lowest scattering, 255=highest scattering) map of the discto allow visual inspection and selection of regions of interest forintegrated backscatter (IB) analysis. Radio frequency (RF) ultrasonicdata are stored in a raster scan format and analyzed with customsoftware. Segments of the RF lines are gated for integrated backscatteranalysis to encompass the front and back surfaces of the nitrocellulosedisc. The data are multiplied by a rectangular window and their powerspectra are determined by fast-Fourier transformation. The power spectrafrom the specimens referenced to the power spectrum returned from anear-perfect steel planar reflector and the frequency-dependentbackscatter transfer function across the useful bandwidth of thetransducer (30 to 60 MHz) are computed and expressed in decibelsrelative to acoustic scattering from the near perfect steel platereflector (Wong et al., Ultrasound in Med & Biol. 1993; 19: 365-374).Integrated backscatter is computed as the average of thefrequency-dependent backscatter transfer function across the usefulbandwidth of the transducer.

Biotinylated, anti-D-dimer F_((ab)) fragment is specifically bound tothe central region of the D-dimer spotted discs and crosslinked byavidin through its biotin moiety. As in previous examples, biotinylatedperfluorocarbon emulsion specifically binds to the antibody boundavidin; whereas, the nonspecific binding of the control emulsion have nobinding and are not detected acoustically. IB of the biotinylatedemulsion coated nitrocellulose (−18.0±0.2 dB) was greater by 4.6±0.1 dB(p<0.05) than that from the control disc (−22.6±0.1 dB) over the 30 to60 MHz frequency range. The frequency-dependent variation in apparentbackscatter transfer function (mean±SEM) of the biotinylated and controlemulsion discs are presented in FIG. 6. A smooth and consistentlygreater acoustic response is noted across the frequency spectrum due tothe bound biotinylated emulsion. These data confirm and extend thefindings of Example 4 with avidin alone, demonstrating that biotinylatedperfluorocarbon emulsion bound through a specific, targeting ligandsystem can significantly enhance the acoustic backscatter of a solidsupport surface.

EXAMPLE 6

Biotinylated perfluorocarbon emulsion (250 nm diameter) is specificallytargeted to avidin conjugated to nitrocellulose discs and insonified atclinically relevant frequencies (5 to 15 MHz) and significantlyincreases the acoustic backscatter of the membrane. Briefly,nitrocellulose membranes (S+S NC™, Schleicher & Schuell, Keane, N.H.)are conjugated to avidin using a diaminohexane (Sigma Chemical Co., St.Louis, Mo.) spacer and glutaraldehyde (Sigma Chemical Co., St. Louis,Mo.) activation as described by Másson et al. (Electrophoresis 1993, 14,860-865). Nitrocellulose discs (2 cm diameter) are soaked in 2.5%diaminohexane dissolved in deionized water for 60 minutes with constant,slow rotary agitation. Membranes are transferred to and washed with 1Macetic acid for 6-7 hours then transferred for continued washing indeionized water for at least 18 additional hours with constantagitation. The membranes are placed in 1% glutaraldehyde in 0.1M sodiumbicarbonate buffer, pH 10.0 for 15 minutes. After glutaraldehydeactivation is complete, the membranes are washed with continuedagitation for three hours. The nitrocellulose membranes stored and driedat 4° C. until use; storage does not exceed three days.

Fifty (50) μl of avidin (250 μg) are spotted dropwise upon the center ofa nitrocellulose membrane with a microliter syringe and allowed to dry.Each membrane is washed with 0.1% Tween-20 (Sigma Chemical Co., St.Louis, Mo.) in phosphate buffered saline (PBS) then placed in 3% bovineserum albumin (BSA, crystallized, Sigma Chemical Company, St. Louis,Mo.) dissolved PBS-0.1% Tween-20 for 20 minutes to blockade nonspecificprotein binding sites around the periphery of the disc. After the BSAblockade, each disc is washed with PBS and placed in 300 μl of eitherbiotinylated or control perfluorocarbon emulsions suspended in 4 ml PBSfor 20 minutes with mild, rotary agitation. The unbound emulsion isremoved with washes of PBS. Each disc is reexposed to avidin, washedwith PBS, reexposed to control or biotinylated perfluorocarbon emulsionand rewashed with PBS as previously described. The nitrocellulose discsare stored in PBS at 4° C. until imaged with the acoustic microscope.

For acoustic microscopic imaging, each nitrocellulose disc is placedflat above a polished stainless steel plate in a polystyrene holder witha 2 cm×2 cm central window removed. The mounted specimen is immersedinto PBS at ambient temperature for ultrasonic insonification. A customdesigned acoustic microscope, utilizing a 10 MHz (nominal frequency)broadband, focused, piezoelectric delay-line transducer (½ inchdiameter, 2 inch focal length, Model V311, Panametrics Co., Waltham,Mass.) operated in the pulse-echo mode is utilized for insonification.Backscattered radio frequency (RF) data is collected and digitized at500 megasamples per second utilizing a Tektronix DSA 601 digitizingoscilloscope (Beaverton, Oreg.) with 8-bit resolution. A variable gainsystem is used to increase the effective dynamic range of thisdigitizer. Radio frequency data are acquired from approximately 100independent sites from each region of interest with 250 micron lateralstep resolution.

A radio frequency peak-detected scan of the data is converted into agray scale (0=lowest scattering, 255=highest scattering) map of the discto allow visual inspection and selection of regions of interest forintegrated backscatter analysis. Radio frequency ultrasonic data arestored in a raster scan format and analyzed with custom software.Segments of the RF lines are gated for integrated backscatter analysisto encompass the front and back surfaces of the nitrocellulose disc. Thedata are multiplied by a rectangular window and their power spectra aredetermined by fast-Fourier transformation. The power spectra from thespecimens referenced to the power spectrum returned from a near-perfectsteel planar reflector and the frequency-dependent backscatter transferfunction across the useful bandwidth of the transducer (5 to 15 MHz) arecomputed and expressed in decibels relative to acoustic scattering fromthe near perfect steel plate reflector (Wong et al., Ultrasound in Med &Biol. 1993; 19: 365-374). Integrated backscatter (IB) is computed as theaverage of the frequency-dependent backscatter transfer function acrossthe useful bandwidth of the transducer.

Discs incubated with biotinylated perfluorocarbon emulsion have centralregions with high acoustic scattering relative to the peripheral regionsof the same disc or the central regions of the control emulsion disc.Nitrocellulose discs incubated with the control emulsion have no highscattering regions. IB of the biotinylated emulsion coatednitrocellulose (0.5±0.5 dB) was greater by 9.6±0.1 dB (8-fold) (p<0.05)than that from the control disc (−9.2±0.5 dB) over the 5 to 15 MHzfrequency range. The frequency-dependent variation in apparentbackscatter transfer function (mean±SEM) of the biotinylated and controlemulsion discs are presented in FIG. 7. A smooth and consistentlygreater acoustic response is noted across the frequency spectrum due tothe bound biotinylated emulsion. These data confirm and extend thefindings of Examples 4 and 5 with avidin and D-dimer, demonstrating thatbiotinylated perfluorocarbon emulsion bound through a specific,targeting ligand system can significantly enhance the acousticbackscatter of a solid support surface and that this improved acousticbackscatter is detected at low, clinically useful ultrasonic frequencies(5 to 15 MHz) as well as high frequencies (30 to 60 MHz).

EXAMPLE 7

Biotinylated perfluorocarbon contrast, approximately 3000 nm diameter,is specifically targeted to avidin conjugated to nitrocellulose discsand insonified at clinically relevant frequencies (at least 5 to 15 MHzbandinidth). Briefly, nitrocellulose membranes (S+S NC™, Schleicher &Schuell, Keane, N.H.) are conjugated to avidin using a diaminohexane(Sigma Chemical Co., St. Louis, Mo.) spacer and glutaraldehyde (SigmaChemical Co., St. Louis, Mo.) activation as described by Masson et al.(Electrophoresis 1993, 14, 860-865). Nitrocellulose discs (2 cmdiameter) are soaked in 2.5% diaminohexane dissolved in deionized waterfor 60 minutes with constant, slow rotary agitation. Membranes aretransferred to and washed with 1M acetic acid for 6-7 hours thentransferred for continued washing in deionized water for at least 18additional hours with constant agitation. The membranes are placed in 1%glutaraldehyde in 0.1M sodium bicarbonate buffer, pH 10.0 for minutes.After glutaraldehyde activation is complete, the membranes are washedwith continued agitation for three hours. The nitrocellulose membranesstored and dried at 4° C. until use; storage does not exceed three days.

Fifty (50) μl of avidin (250 μg) are spotted dropwise upon the center oftwo of four membranes with a microliter syringe and allowed to dry. Eachmembrane is washed with 0.1% Tween-20 (Sigma Chemical Co., St. Louis,Mo.) in phosphate buffered saline (PBS) then placed in 3% bovine serumalbumin (BSA, crystallized, Sigma Chemical Company, St. Louis, Mo.)dissolved PBS-0.1% Tween-20 for 20 minutes to blockade nonspecificprotein binding sites around the periphery of the disc. After the BSAblockade, each disc is washed with PBS and placed in 300 μl of eitherbiotinylated or control perfluorocarbon emulsions, approximately 3000 nmparticle size, suspended in 4 ml PBS for 20 minutes with mild, rotaryagitation. The unbound emulsion is removed with washes of PBS. Each discis reexposed to avidin, washed with PBS, exposed to perfluorocarbonemulsion and rewashed with PBS as previously described. Thenitrocellulose discs are stored in PBS at 4° C. until imaged with theacoustic microscope.

For acoustic microscope imaging, each nitrocellulose disc is placed flatabove a polished stainless steel plate in a polystyrene holder with a 2cm×2 cm central window removed. The mounted specimen is immersed intoPBS at ambient temperature for ultrasonic insonification. A customdesigned acoustic microscope, utilizing a broadband 10 MHz (nominalfrequency) focused, piezoelectric delay-line transducer (½ inchdiameter, 2 inch focal length, Model V311, Panametrics Co., Waltham,Mass.) operated in the pulse-echo mode is utilized for insonification.Backscattered radio frequency (RF) data is collected and digitized at500 megasamples per second utilizing a Tektronic DSA 601 digitizingoscilloscope (Beaverton, Oreg.) with 8-bit resolution. A variable gainsystem is used to increase the effective dynamic range of thisdigitizer. Radio frequency data are acquired from approximately 100independent sites from each region of interest with 250 micron lateralstep resolution.

A radio frequency peak-detected scan of the data is converted into agray scale (0=lowest scattering, 255=highest scattering) map of the discto allow visual inspection and selection of regions of interest forintegrated backscatter analysis. Radio frequency ultrasonic data arestored in a raster scan format and analyzed with custom software.Segments of the RF lines are gated for integrated backscatter (IB)analysis to encompass the front and back surfaces of the nitrocellulosedisc. The data are multiplied by a rectangular window and their powerspectra are determined by fast-Fourier transformation. The power spectrafrom the specimens referenced to the power spectrum returned from anear-perfect steel planar reflector and the frequency-dependentbackscatter transfer function across the useful bandwidth of thetransducer (5 to 15 MHz) are computed and expressed in decibels relativeto acoustic scattering from the near perfect steel plate reflector (Wonget al., Ultrasound in Med & Biol. 1993; 19: 365-374). Integratedbackscatter is computed as the average of the frequency-dependentbackscatter transfer function across the useful bandwidth of thetransducer.

Discs incubated with biotinylated perfluorocarbon emulsion have centralregions with high acoustic scattering relative to the peripheral regionsof the same disc and central regions of the control disc. Nitrocellulosediscs incubated with the control emulsion have no central highscattering regions and no differences in acoustic character are detectedbetween the central and peripheral regions of the disc. IB of thebiotinylated emulsion coated nitrocellulose (−2.4±0.7 dB) was greater by8.8±0.3 dB (approximately 8-fold (p<0.05)) than that from the controldisc (−11.2±0.4 dB) over the 5 to 15 MHz frequency range. Thefrequency-dependent variation in apparent backscatter transfer function(mean±SEM) of the biotinylated and control emulsion discs are presentedin FIG. 8. A smooth and consistently greater acoustic response is notedacross the frequency spectrum due to the bound biotinylated emulsion.These data confirm and extend the findings of Examples 4, 5 and 6 withavidin and D-dimer, demonstrating that biotinylated perfluorocarbonemulsions with large particle sizes can be bound through a specific,targeting ligand system and significantly enhance the acousticbackscatter of a solid support surface. This improved acousticbackscatter is detected at clinically relevant ultrasonic frequencies, 5to 15 MHZ.

EXAMPLE 8

Biotinylated perfluorocarbon emulsion is targeted to a plasma thrombiusing biotinylated antifibrin monoclonal antibodies (NIB 1H10; Tymkewyczet al. 1993. Blood Coagulation and Fibrinolysis 4:211-221) and avidin.In a representative study (1 of 5), whole porcine blood is obtained andanticoagulated (9:1, v/v) with sterile sodium citrate. Blood iscentrifuged at 1500 RPM at room temperature and the plasma fraction isobtained and stored at 4° C. Two porcine plasma thrombi are produced bycombining plasma, 100 mM calcium chloride (3:1 v/v) and 2-5 U thrombinin a plastic tube through which 5-0 Vicryl suture is passed. Thrombi areallowed to coagulate at room temperature.

One thrombus is incubated with 150 μg antifibrin monoclonal antibody in10 ml PBS with 1% bovine serum albumin (BSA) for two hours and a secondcontrol thrombus is incubated in PBS with 1% BSA. The antibody treatedthrombus is then incubated with 0.5 mg avidin in 10 ml PBS with 1% BSAfor 30 minutes. The control thrombus remains in PBS with 1% BSA. Boththrombi are washed extensively with PBS. Each thrombus is incubated with300 μgl/10 ml PBS of either biotinylated or control emulsion for 30minutes. All thrombi are reexposed to emulsion twice to ensure uniformcoverage and ultrasonically insonified (FIG. 9). Ultrasonic imaging isperformed using a 7.5 MHz focused, linear phased array transducer and aHewlett Packard Sonos 2500 Imaging System (Hewlett Packard, Inc.,Andover, Mass.). All ultrasonic recordings are produced with fixed gain,compensation and time-gain compensation levels and are recorded on toSVHS videotape for subsequent image analysis. Average pixel grayscaleover an extensive region of interest was sampled for 21 independentfreeze-frame images for each thrombus using NIH Image 1.47 (NationalInstitutes of Health; FIG. 10). The biotinylated perfluorocarbonemulsion is found to provide a marked acoustic enhancement of thesurface. Average pixel grayscale levels of the biotinylated emulsionthrombus are 79.5±2.5 whereas the brightness of the control was markedlyless (34.8±2.2, p<0.05). These results demonstrate the ability ofbiotinylated perfluorocarbon emulsion to target and acoustically enhancea biological tissue (i.e. thrombus) in vitro.

EXAMPLE 9

Biotinylated perfluorocarbon emulsion is targeted via biotinylatedantifibrin antibodies (NIB5F3 and NIB1H10 Tymkewycz et al. 1993. BloodCoagulation and Fibrinolysis 4:211-221) to an isolated femoral arterythrombus in six mongrel dogs. A mongrel dog is anesthetized with sodiumpentobarbital induction and halothane anesthesia. The right femoralartery and all branches are isolated at the level of the saphenousbranch. A silver plated copper wire attached to a 22 ga. right angledneedle point, insulated with plastic tubing (polyethylene P-240), isinserted into the femoral artery and secured with 4-0 Prolene suture. Acurrent of 200-400 μA is applied for up to two hours. Thrombus formationis monitored with continuous wave doppler and discontinued after anapproximately 50% increase in circulation velocity is noted distal tothe electrical injury. Adventitial discoloration secondary to thecurrent is appreciated proximal to the entry point of the wire. A 20 ga.catheter is inserted into a proximal branch of the femoral artery andsecured with 4-0 silk suture. A pressurized 0.9% NaCl drip is attachedthrough a three-way stopcock to the catheter. Blood flow into theisolated segment is disrupted by proximal snare ligature. Excess bloodis flushed from the arterial segment to inhibit further thrombusformation by infusion of saline for 15 minutes. The distal drainingbranches of the femoral artery are ligated or snared with suture.Biotinylated antifibrin monoclonal antibody (50 μg/1.0 ml PBS) isinjected via the catheter and flushed with a few drops of saline. Theantibody is allowed to incubate for one hour then the snare ligaturedistal to the wire insertion is released and excess antibody is flushedthrough with saline for five minutes. The distal femoral artery isreoccluded and avidin (250 μg/1.0 ml PBS) is infused and incubates for30 minutes. The distal ligature is again released and excess avidin isflushed through with saline for five minutes. The distal ligature isreestablished and biotinylated perfluorocarbon emulsion is infused andincubates for 30 minutes. After the initial exposure of the thrombus tothe emulsion, the unbound emulsion is washed through with saline.Thrombi are each exposed to avidin and biotinylated perfluorocarbonemulsion as described above. In three animals, the contra lateral arteryis also isolated, partially occluded with electrically induced thrombiand exposed to a control perfluorocarbon emulsion analagous to theadministration of biotinylated emulsion described above. Femoralarteries exposed to either control or biotinylated perfluorocarbonemulsion are ultrasonically imaged at 7.5 MHz with a focused, linearphased array transducer and a clinical Hewlett-Packard Sonos 2500Ultrasonic Imaging System before and after contrast administration.Acutely formed thrombi, both control and contrast targeted, are notultrasonically appreciated. For 6 of 6 femoral arteries, partiallyocclusive thrombi are markedly enhanced using the antifibrin targetedbiotinylated perfluorocarbon contrast. In 3 of 3 femoral arteriesthrombi, exposure to the control perfluorocarbon emulsion does notaccentuate their acoustic reflectivity and these thrombi remainultrasonically undetectable. FIG. 11 reveals a representative example ofa femoral artery site of thrombus formation after electrical inductionbefore and after exposure to antifibrin antibody and biotinylatedcontrast. In the pre-contrast image, the femoral artery is observed witha bright echogenic wire point anode protruding into the lumen but nothrombus is appreciated. After treatment with the biotinylated contrastemulsion, a large partially occluded thrombus is clearly noted by theenhanced acoustic reflectivity (FIG. 11). Again, no thrombus isappreciated in the control artery before or after exposure to controlemulsion. These results demonstrate the concept of using boundperfluorocarbon emulsion to acoustically enhance biological surfaces,such as thrombotic tissue, in vivo to enable detection with acommercially available ultrasound imaging system.

EXAMPLE 10

Biotinylated perfluorocarbon emulsion, approximately 250 nm diameter, istargeted to prostatic carcinoma using monoclonal antibodies specific forprostate specific antigen (PSA) and are acoustically detected usingpolar, high frequency, high resolution acoustic microscopy.Representative examples of human prostatic carcinoma tissues areroutinely processed by immersion fixation in 10% neutral bufferedformalin and embedded in paraffin. Twenty micron sections are preparedfor acoustic microscopy; 5 micron sections are used for optical studies.All histologic sections are mounted on acid cleaned glass slides thathave been coated with poly-L-lysine. All mounted sections are heated at55° C. for 1 hour in an oven.

Prior to immunostaining, all sections are dewaxed in three changes ofAmericlear, and dehydrated in successive changes of 95% and 100%ethanol. Endogenous peroxidase activity is blocked only in sectionsprepared for optical studies by immersion in absolute methanolcontaining 0.6% (v/v) hydrogen peroxide for 30 minutes. These and allsections for acoustic microscopy are then rehydrated through gradedethanols and distilled water and placed in isotonic PBS (pH 7.4). Allsections are incubated with target specific monoclonal antibodies.Prostate sections are incubated with anti-PSA primary monoclonalantibodies per the recommendations of the vendor for 18 hours at 4° C.in moisture chambers. After primary incubation, sections are rinsed inisotonic PBS, then overlain with a polyclonal biotinyl-horse anti-mouseimmunoglobin (VectaStain Elite Kits, Vector Laboratories, Burlingame,Calif.) for 1 hour at room temperature. After rinsing in PBS, a 30micron section is prepared for acoustic microscopy. A section for lightmicroscopy (5 micron) is incubated with avidin-biotin-peroxidase complex(VectaStain Elite Kit, Vector Lab) for 1 hour at room temperature. Thissection is rinsed in phosphate buffer (pH 7.6) and immersed in asolution of 3,3′-diaminobenzidine tetrahydrochloride (Sigma Chemicals,St. Louis, Mo.; 0.5 mg/ml in phosphate buffer, pH 7.6, containing 0.003%[v/v] hydrogen peroxide) for approximately ten minutes. The chromogenicprecipitate is optically enhanced by brief immersion of stained sectionsin 0.125% (w/v) osmium tetroxide. The section is then rinsed in tapwater, counterstained in Harris' hematoxylin, dehydrated in gradedethanols and Americlear, and mounted in a synthetic mounting medium.

After the second biotinylated antibody is incubated and washed, slidesfor acoustic microscopy are incubated in avidin (1.0 mg/˜20 cc PBS)using a bath on a rotating table for 30 min. Excess avidin is washedaway with isotonic PBS buffer, pH 7.4-7.5 in three minute washes. Slidesare incubated with biotinylated or control perfluorocarbon emulsion fortwenty minutes (0.5 cc/˜20.0 ml PBS), washed briefly with isotonic PBS3× for 5 minutes each and reincubated with avidin (1.0 mg/˜20 cc) for 15minutes. Excess avidin is rinsed off with three, 5 min. washes in PBS.The slide is then reincubated at above concentrations with biotinylatedor control perfluorocarbon emulsion for 20 minutes. Unbound emulsion iswashed away in three changes of PBS (5 minutes each) and the slides aretransferred to the acoustic microscope for analysis.

The mounted specimens are each immersed into isotonic, phosphatebuffered saline at room temperature for ultrasonic insonification. Acustom designed acoustic microscope is used to collect ultrasonic data.The microscope consists of 50 MHz broadband, focused, piezoelectricdelay-line transducer (¼ inch diameter, ½ inch focal length, 62 micronbeam diameter, Model V390, Panametrics Co., Waltham, Mass.) operated inthe pulse-echo mode. A Tektronix DSA 601 digitizing oscilloscope(Beaverton, Oreg.) is used to digitize 35 degree polar backscatteredradio frequency (rf) data at 500 megasamples per second with 8-bitresolution. A variable gain system is used to increase the effectivedynamic range of this digitizer. Radio frequency data is acquired fromapproximately 100 independent sites from each specimen with 50 micronlateral step resolution.

The rf data is stored in a low resolution raster scan format andanalyzed with custom software. Segments of the rf lines are gated forintegrated backscatter analysis to encompass the front surface (i.e.excluding the back wall). The gated data are multiplied by a Hammingwindow and their power spectra are determined by fast-Fouriertransformation. Power spectra within a tissue section are compareddirectly without reference to a steel plate. Integrated backscatter (IB)is computed from the average of the frequency-dependent backscattertransfer function across the useful bandwidth of the transducer (30 to55 MHz). Immunostained tissues are reviewed using a Nikon Optiphot-2microscope for regions of PSA positive staining and the acousticcharacteristics are compared.

The net change in the apparent backscatter transfer function between thenormal prostatic stroma and carcinomatous regions are clearly increasedin sections treated with PSA targeted biotinylated versus the controlperfluorocarbon emulsion across the frequency spectrum (30 to 55 MHz;FIG. 12). Biotinylated perfluorocarbon emulsion increases (p<0.05) theintegrated backscatter from regions of prostatic cancer (47.17±dB)versus normal stromal (40.79±1.18 dB) by 6.38 dB (approximately 4-fold).In the control tissue sections, the integrated backscatter from theregion of prostatic carcinoma (39.63±1.63 dB) was greater (p<0.05) thanthat from the normal stromal areas (36.13±2.17 dB) by approximately 3.5dB (2-fold), reflecting inherent differences in acoustic characterbetween normal and cancerous prostatic tissue. However, the targetedbiotinylated perfluorocarbon emulsion amplified (p<0.05) these inherentdifferences by approximately 2-fold (2.87 dB; FIG. 13). These resultsclearly demonstrate the ability of site-targeted biotinylatedperfluorocarbon emulsion to specifically enhance acoustic detection ofprostate cancer in vitro.

EXAMPLE 11

Biotinylated perfluorocarbon emulsion, approximately 250 nm diameter, istargeted to ovarian carcinoma using monoclonal antibodies specific forOC-125 antigen and are acoustically detected using polar, highfrequency, high resolution acoustic microscopy. Representative examplesof human ovarian, carcinoma tissues are routinely processed by immersionfixation in 10% neutral buffered formalin and embedded in paraffin.Twenty micron sections are prepared for acoustic microscopy; 5 micronsections are used for optical studies. All histologic sections aremounted on acid cleaned glass slides that have been coated withpoly-L-lysine. All mounted sections are heated to 55° C. for 1 hour inan oven.

Prior to immunostaining, all sections are dewaxed in three changes ofAmericlear, and dehydrated in successive changes of 95% and 100%ethanol. Endogenous peroxidase activity is blocked only in sectionsprepared for optical studies by immersion in absolute methanolcontaining 0.6% (v/v) hydrogen peroxide for 30 minutes. These and allsections for acoustic microscopy are then rehydrated through gradedethanols and distilled water and placed in isotonic PBS (pH 7.4). Allsections are incubated with target specific monoclonal antibodies.

Ovarian sections are incubated with anti-OC-125 primary monoclonalantibodies per the recommendations of the vendor for 18 hours at 4° C.in moisture chambers. After primary incubation, sections are rinsed inisotonic PBS, then overlain with a polyclonal biotinyl-horse anti-mouseimmunoglobin (VectaStain Elite Kits, Vector Laboratories, Burlingame,Calif.) for 1 hour at room temperature. After rinsing in PBS, duplicate30 micron sections are prepared for acoustic microscopy. Sections forlight microscopy (5 micron) are incubated with avidin-biotin-peroxidasecomplex (VectaStain Elite Kit, Vector Lab) for 1 hour at roomtemperature. Sections are rinsed in phosphate buffer (pH 7.6) andimmersed in a solution at 3,3′-diaminobenzidine tetrahydrochloride(Sigma Chemicals, St. Louis, Mo.; 0.5 mg/ml in phosphate buffer, pH 7.6,containing 0.0003% [v/v] hydrogen peroxide) for approximately tenminutes. The chromogenic precipitate is optically enhanced by briefimmersion of stained sections in 0.125% (w/v) osmium tetroxide. Sectionsare then rinsed in tap water, counterstained in Harris' hematoxylin,dehydrated in graded ethanols and Americlear, and mounted in a syntheticmounting medium.

After the second biotinylated antibody is incubated and washed, slidesare incubated in avidin (1.0 mg/˜20 cc PBS) using a bath on a rotatingtable for 30 min. Excess avidin is washed away with isotonic PBS buffer,pH 7.4-7.5 in three 5 minute washes. The prepared slides are incubatedwith biotinylated or control perfluorocarbon emulsion for twenty minutes(0.5 cc/˜20.0 ml PBS), washed briefly with isotonic PBS 3× for 5 minuteseach and rewashed in avidin (1.0 mg/˜20 cc) for 15 minutes. Excessavidin is rinsed off with three, 5 min. washes in PBS. Slides are thenreincubated at above concentrations with biotinylated or controlperfluorocarbon emulsion for 20 minutes. Unbound emulsion is washed awayin three changes of PBS (5 minutes each) and the slides are transferredto the acoustic microscope for analysis.

The mounted specimens are each immersed into isotonic, phosphatebuffered saline at room temperature for ultrasonic insonification. Acustom designed acoustic microscope is used to collect ultrasonic data.The microscope consists of 50 MHz broadband, focused, piezoelectricdelay-line transducer (¼ inch diameter, ½ inch focal length, 62 micronbeam diameter, Model V390, Panametrics Co., Waltham, Mass.) operated inthe pulse-echo mode. A Tektronix DSA 601 digitizing oscilloscope(Beaverton, Oreg.) is used to digitize 35 degree polar backscatteredradio frequency (rf) data at 500 megasamples per second with 8-bitresolution. A variable gain system is used to increase the effectivedynamic range of this digitizer. Radio frequency data is acquired fromapproximately 100 independent sites from each specimen with 50 micronlateral step resolution.

The rf data are stored in a low resolution raster scan format andanalyzed with custom software. Segments of the rf lines are gated forintegrated backscatter analysis to encompass the front surface (i.e.excluding the back wall). The gated data are multiplied by a Hammingwindow and their power spectra are determined by fast-Fouriertransformation. Integrated backscatter is computed from the average ofthe frequency-dependent backscatter transfer function across the usefulbandwidth of the transducer (30 to 55 MHz). The power spectra from thespecimens are referenced to the power spectrum returned from a glassmicroscope slide. IB is expressed in decibels relative to the scatteringfrom the glass slide. Immunostained tissues are reviewed using a NikonOptiphot-2 microscope for regions of PSA positive staining and theacoustic characteristics are compared.

The net change in the apparent backscatter transfer function between thenormal ovarian stroma and carcinomatous regions are clearly increased insections treated with OC125 targeted biotinylated versus the controlperfluorocarbon emulsion across the frequency spectrum (30 to 55 MHz;FIG. 14). Biotinylated perfluorocarbon emulsion increases (p<0.05) theintegrated backscatter from regions of ovarian cancer (−28.19±1.39 dB)versus normal stromal (−38.75±0.84 dB) by 10.57 dB (greater than8-fold). In the control tissue sections, the integrated backscatter fromthe region of ovarian carcinoma (−33.49±0.86 dB) was greater (p<0.05)than the normal stromal areas (−40.21±0.61 dB), approximately 6.72 dB(4-fold), reflecting inherent differences in acoustic character betweennormal and cancerous ovarian tissue. However, the targeted biotinylatedperfluorocarbon emulsion amplified (p<0.05) these inherent differencesby approximately 2-fold (3.84 dB; FIG. 15). These results clearlydemonstrate the ability of site-targeted biotinylated perfluorocarbonemulsion to specifically enhance acoustic detection of ovarian cancer invitro.

EXAMPLE 12

Biotinylated perfluorocarbon emulsion, approximately 250 nm diameter, istargeted to the epithelial capsule of tonsil using monoclonal antibodiesspecific for cytokeratin, CD-20, and BCL-2 antigens and are acousticallydetected using polar, high frequency, high resolution acousticmicroscopy. Representative examples of human tonsil are routinelyprocessed by immersion fixation in 10% neutral buffered formalin andembedded in paraffin. Twenty micron sections are prepared for acousticmicroscopy; a 5 micron section is used for optical studies. Allhistologic sections are mounted on acid cleaned glass slides that havebeen coated with poly-L-lysine. All mounted sections are heated at 55°C. for 1 hour in an oven.

Prior to immunostaining, all sections are dewaxed in three changes ofAmericlear, and dehydrated in successive changes of 95% and 100%ethanol. Endogenous peroxidase activity is blocked only in sectionsprepared for optical studies by immersion in absolute methanolcontaining 0.6% (v/v) hydrogen peroxide for 30 minutes. These and allsections for acoustic microscopy are then rehydrated through gradedethanols and distilled water and placed in isotonic PBS (pH 7.4). Allsections are incubated with target specific monoclonal antibodies.

Tonsil sections are incubated with a mixture of anti-CD-20, BCL-2, andcytokeratin primary monoclonal antibodies per the recommendations of thevendor for 18 hours at 4° C. in moisture chambers. After primaryincubation, sections are rinsed in isotonic PBS, then overlain with apolyclonal biotinyl-horse anti-mouse immunoglobin (VectaStain EliteKits, Vector Laboratories, Burlingame, Calif.) for 1 hour at roomtemperature. After rinsing in PBS, duplicate 30 micron sections areprepared for acoustic microscopy. Sections for light microscopy (5micron) are incubated with avidin-biotin-peroxidase complex (VectaStainElite Kit, Vector Lab) for 1 hour at room temperature. Sections arerinsed in phosphate buffer (pH 7.6) and immersed in a solution of3,3′-diaminobenzidine tetrahydrochloride (Sigma Chemicals, St. Louis,Mo.; 0.5 mg/ml in phosphate buffer, pH 7.6, containing 0.003% [v/v]hydrogen peroxide) for approximately ten minutes. The chromogenicprecipitate are optically enhanced by brief immersion of stainedsections in 0.125% (w/v) osmium tetroxide. Sections are then rinsed intap water, counterstained in Harris' hematoxylin, dehydrated in gradedethanols and Americlear, and mounted in a synthetic mounting medium.

After the second biotinylated antibody is incubated and washed, oneslide is incubated in avidin (1.0 mg/˜20 cc PBS) using a bath on arotating table for 30 min. Excess avidin is washed away with isotonicPBS buffer, pH 7.4-7.5 in three 5 minute washes. The prepared slide isincubated with biotinylated perfluorocarbon emulsion for twenty minutes(0.5 cc/˜20.0 ml PBS), washed briefly with isotonic PBS 3× for 5 minuteseach and rewashed in avidin (1.0 mg/˜20 cc) for 15 minutes. Excessavidin is rinsed off with three, 5 minute washes in PBS. The slides arereincubated at above concentrations with biotinylated perfluorocarbonemulsion for 20 minutes. Unbound emulsion is washed away in threechanges of PBS (5 minutes each) and the slide is transferred to theacoustic microscope for analysis.

The mounted specimen is immersed into isotonic, phosphate bufferedsaline at room temperature for ultrasonic insonification. A customdesigned acoustic microscope is used to collect ultrasonic data. Themicroscope consists of a 50 MHz broadband, focused, piezoelectricdelay-line transducer (¼ inch diameter, ½ inch focal length, 62 micronbeam diameter, Model V390, Panametrics Co., Waltham, Mass.) operated inthe pulse-echo mode. A Tektronix DSA 601 digitizing oscilloscope(Beaverton, Oreg.) is used to digitize 35 degree polar backscatteredradio frequency (rf) data at 500 megasamples per second with 8 bitresolution. A variable gain system is used to increase the effectivedynamic range of this digitizer. Radio frequency data are collected fromthe entire specimen and a peak detected image is created of the sectionand compared with the immunostained tissue image.

Immunostained tissue is examined and imaged using a Nikon Optiphot-2microscope with a Javlin Chromachip II camera attachment. Images arerouted through a Panasonic digital mixer model WJ-AVE5 to Panasonic SVHSvideo recorders, models AG-1960 or AG-1970 and displayed upon an SonyTrinitron monitor. Images are captured using NuVista software(Truevision, Inc., Indianapolis, Ind. 46256) executing on a MacintoshLCIII microcomputer.

FIG. 16 compares tonsil acoustically imaged as a radio frequency peakdetected scan at 100 micron lateral step resolution (a) with anoptically imaged section immunostained with horseradish peroxidase (b).The epithelial capsule targeted by a mixture of anti-cytokeratinantibodies is distinctly stained with horseradish peroxidase andhomologous regions in the acoustic image are “brightened” by thetargeted biotinylated acoustic contrast. In FIG. 17 the radio frequencypeak detected acoustic image at 100 micron step resolution (a) isenhanced to 50 micron lateral step resolution. The targeted biotinylatedperfluorocarbon contrast is clearly seen acoustically enhancing theepithelial rim of the tonsil, analogous to the optical immunostainedimage. This example clearly demonstrates the fidelity of biotinylatedperfluorocarbon contrast targeting for enhanced acoustic contrast oftissues, such as lymph nodes.

EXAMPLE 13 A Method to Chemically Couple Antibody to PerfluorocarbonEmulsion Particle

Preparation of Emulsion: The perfluorocarbon nanoparticle contrast agentis produced by incorporating1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-4-(p-maleimidophenyl)butyramide(MPB-PE) into the outer lipid monolayer of the emulsion. The emulsion iscomprised of perfluorodichlorooctane, safflower oil, a surfactantco-mixture and glycerin. The surfactant co-mixture includes lecithin,cholesterol and MPB-PE which is dissolved in chloroform. Thechloroform-lipid mixture is evaporated under reduced pressure, dried ina 50° C. vacuum oven overnight and dispersed into water by sonication.The suspension is transferred into a blender cup withperfluorodichloroocatane, safflower oil and distilled, deionized waterand emulsified for 30 to 60 seconds. The pre-emulsified mixture istransferred to a microemulsifier and continuously processed at 10,000PSI for three minutes. The completed emulsion is vialed, blanketed withnitrogen and sealed with stopper crimp seal until use. A negativecontrol emulsion is prepared identically, except a nonderivatizedphosphatidylethanolamine is substituted into the surfactant co-mixture.Particle sizes are determined in triplicate at 30° C. with a laser lightscatter submicron particle size analyzer.

Preparation and Isolation of Tissue Factor F(ab)′ Fragments: F(ab)′fragments of a specific antibody are generated and isolated using animmunopure F(ab)′ preparation kit (Pierce, Rockford, Ill.). Typically,IgG antibody is dialyzed into 20 mM phosphate/10 mM EDTA buffer (pH7.0), concentrated to 20 mg/ml and digested by immobilized papain.Solubilized F9ab)′ are purified from Fc fragments and undigested IgGprotein using a protein A column. F(ab)′ fragments are purified fromexcess cysteine using a G25-15-column and deoxygenated phosphate buffer(pH 6.7). Fraction identity is confirmed by routine SDS-PAGE procedures.

Conjugation of Tissue Factor F(ab)′ With MPB-PE Derivatized Emulsion:F(ab)′ fractions are pooled and combined with the MPB-PE derivatizedemulsion (0.01 to 5.0 mg F(ab)′/ml of emulsion, preferably 1 to 2 mgF(ab)′/ml of emulsion). The mixture is adjusted to pH 6.7, sealed undernitrogen and allowed to react overnight at ambient temperatures withgentle, continuous mixing. The mixture may be subsequently dialyzed witha 300,000 MWCO Spectra/Por DispoDialyzer (Laguna Hills, Calif.) against10 mM phosphate buffer (pH 7.2) to remove unconjugated F(ab)′ fragments.The final emulsion is vialed under nitrogen and stored at 4° C. untiluse.

EXAMPLE 14 In vitro Targeting of Fibrin-Rich Plasma Thrombi Using AFibrin-Targeted, One-Step Acoustic Contrast System

Whole blood was obtained fresh and anticoagulated (9:1, v/v) withsterile sodium citrate. In a series of trials, plasma clots (9) wereproduced by combining plasma and 100 mM calcium chloride (3:1, v/v) with5 units of thrombin (Sigma Chemical Company, St. Louis, Mo.) in aplastic tube overlying nitrocellulose membranes. The plasma was allowedto coagulate slowly at room temperature.

Plasma clots were incubated with anti-fibrin (fab) conjugated ornon-conjugated control emulsion contrast using antifibrin monoclonalantibodies (NIB-5F3 or NIB-1H10) (Tymkewycz et al. 1992; Tymkewycz etal. 1993). Half of the clots (5) were incubated individually with 150 μgbiotinylated antifibrin monoclonal antibody in 10 ml PBS with 1% bovineserum albumin, (crystallized, Sigma Chemical Company, St. Louis, Mo.)for two hours; the remaining clots (4) were maintained in PBS with 1%bovine serum albumin. Bovine serum albumin was added during antibodyincubations to minimize nonspecific protein binding to the polystyrenepetri dish walls. The anti-fibrin targeted emulsion was incubated withclots (0.2 ml) for 30 minutes. Control clots were treated similarly witha nontargeted control perfluorocarbon emulsion (0.2 ml) for 30 minutes.The plasma clots on nitrocellulose were insonified using an acousticmicroscope to assess the change in ultrasonic backscattered powerattributable to the control and targeted emulsions.

The microscope consisted of a 50 MHZ broadband, focused, piezoelectricdelay-line transduce (¼ inch diameter, ½ inch focal length, Model V390,Panametrics Co., Waltham, Mass.) operated in the pulse-echo mode. ATektronix DSA 601 digitizing oscilloscope (Beaverton, Oreg.) was used todigitize backscattered radiofrequency data at 500 megasamples per secondwith 8-bit resolution. Radiofrequency data collected from each site wasaveraged 32 times. Averaged radiofrequency data were acquired fromapproximately 400 independent sites with 50 micron lateral stepresolution. The radiofrequency data are stored in a low resolutionraster scan format and analyzed with custom software. Segments of theradiofrequency lines, 500 nsec in duration and encompassing surfacereflection are gated for analysis. The gated data are multiplied by aHamming window and their power spectra determined by fast-Fouriertransformation.

The power spectra from each specimen was referenced to the powerspectrum backscattered from a near-perfect steel plate reflector tocompute the apparent frequency-dependent backscatter transfer function.The backscatter transfer function for the acoustic reflectivity of thesmooth cells, B(f), was expressed in decibels relative to the powerreflected from the steel plate:

B(f)²=10log[V _((f)) ² _(tissue) ]/[V _((f)) ² _(steel plate)]

where V_((f)) ² _(tissue) is the power at selected frequency of thegated rf backscattered from the cells and V_((f)) ² _(steel plate) isthe power at the same frequency of the gated rf backscattered from thesteel plate. Integrated backscatter (IB) was computed from the averageof the frequency-dependent backscatter transfer function over the usefulbandwidth of the transducer (FIG. 18).

EXAMPLE 15 Scanning Electron Micrographs Identification of Tissue FactorEpitopes On Surfaces of Cultured Endothelial Cells Using the One-Step,Tissue Factor Targeted Nanoparticle Emulsion

Porcine aorta smooth muscle cells known to express tissue factor inabundance on their cell surface were cultured onto 12 mm round, glasscover slips and grown for 48 hours at 37° C. under 5% CO2. Fourtreatment groups were prepared: 1) cells alone (untreated control); 2)cells exposed to a nontargeted control emulsion (negative control); 3)cells exposed to anti-tissue factor antibody before treatment withtissue-factor targeted emulsion (blocked control); and 4) cells treatedwith tissue-factor targeted emulsion. All smooth muscle cell cultureswere fixed in 2.5% glutaraldehyde in PBS for one hour, rinsed briefly inPBS and post-fixed in 2% osmium tetroxide for 1 hour. The fixed andwashed cells were dehydrated in ascending concentrations of ethylalcohol, 10 minutes/step: 50%, 70%, 90% and 3×100% followed by three15-minute washes in hexamethyldisilazane (Electron Microscopy Sciences,Forth Worth, Pa.). The glass slips supporting the fixed cells wereplaced on stubs and imaged with a scanning electron microscope.

Only trace nonspecific binding was appreciated in cell cultures treatedwith no (FIG. 19A) or untargeted control emulsion (FIG. 19B). Tissuefactor targeted specifically bound to the aortic smooth muscle cells(FIG. 19C) and this binding could be specifically blocked by anti-tissuefactor antiserum (FIG. 19D). These results demonstrate the ligandspecificity of the targeted perfluorocarbon nonospheres.

EXAMPLE 16 Local Delivery of One-Step Tissue Factor TargetedNanoparticle Emulsion In Balloon-Injured Arteries In Pigs

Briefly, pigs weighing approximately 20 kg were anesthetized withtelazol cocktail (1 ml/23 kg IM) followed by 1-2% isoflurane in oxygen.Both common carotids were exposed aseptically; and the distal portion ofeach, proximal to the bifurcation of the internal carotid artery, wascannulated and connected to a Touhy adapter. The right femoral arterywas exposed, and an 8 French sheath was inserted through which a guidecatheter and 8 mm balloon catheter was directed to each carotid using anexchange wire. The balloon was inflated five times with sequentiallyincreased pressure up to 6 atmospheres for 30 seconds with one minutebetween inflations. Prior to the fifth balloon deflation, heparinized(10 units/ml) saline was infused through the distal Touhy access. As thecatheter was slowly deflated for the final time and withdrawn from thecommon carotid, the residual blood within was flushed from the artery.When the catheter was free of the carotid artery, a proximal snare wasplaced, isolating the carotid segment containing buffer. The anti-tissuefactor targeted nanoparticulate emulsion (0.5 ml) was infused into theright carotid artery and allowed to incubate for 2 hours. After 2 hoursthe formulation was removed, and the artery was washed with 0.9% NaCl.The left carotid artery was exposed to an nonspecific f(ab)-conjugatednanoparticulate emulsion. Vessels were ultrasonically imaged on eachside following the formulation washout (FIG. 20).

Following angioplasty, but prior to application of contrast agent,carotid arterial walls exhibited no evidence of augmented acousticreflectivity secondary to overstretch injury alone. Exposure to thecontrol nonspecific emulsion did not alter the acoustic character of thearterial wall. However, the tissue factor targeted emulsion agentclearly penetrated the vascular tissue and localized to the media whereincreased tissue factor expression was inducted, resulting insubstantial augmentation of acoustic contrast. Acoustic enhancement wasnot circumferentially distributed, but was concentrated over one-half totwo-thirds of the vessel wall where non-circumferential force wasapplied by the angioplasty balloon.

Immunohistochemistry: Vessel segments were fixed in molecular biologyfixative (Streck Laboratories, Omaha, Nebr.), embedded in paraffin, cutat 5 μM, and mounted on gelatin-coated slides. A slide from each vesselwas stained with Verhoeff's van Gieson elastin stain to verify ruptureof the internal elastic lamina as an index of deep vascular injury. Forimmunohistochemistry, the tissue sections were deparaffinized withxylene, hydrated with a descending ethanol series, and rinsed with PBS.Endogenous peroxidase activity was blocked by incubation for 30 minuteswith hydrogen peroxide in methanol. Nonspecific binding of antibody wasblocked by incubation for 45 minutes in PBS containing 1% bovine serumalbumin, 0.3% triton X-100, 10% normal goat serum, and 1% normal pigserum. The sections were incubated overnight at 4° C. with the rabbitanti-pig tissue factor antibody diluted 1:100 in the blocking solution.After rinsing the slides with PBS, the sections were incubated for 45minutes at room temperature with biotinylated goat anti-rabbit IgG(Vector Laboratories) diluted 1:200 with blocking buffer. The sectionswere stained with AEC (HistostainPlus, Zymed) and counterstained withhematoxylin.

Immunohistochemistry revealed a pattern of localization of tissue factorin balloon-injured vessels in the media (arrows) at the site of acousticcontrast enhancement, as well as on the intimal surface associated withinflammatory cells (FIG. 21A; L=lumen). In contrast, control uninjuredvessels exhibited staining primarily in the adventitia and not in thetunica media (FIG. 21B). No obvious dissections of the arterial wallswere observed microscopically, although some mild fenestration of theinternal elastic lamina was noted. These results demonstrate thepresence of tissue factor only in the media of balloon overstretchedarteries. The vessels were acoustically enhanced by the tissue factortargeted, one-step nanoparticulate emulsion, but not by the nontargetedcontrol nanoparticulate emulsion.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A method for enhanced ultrasound imaging of a target tissue in a subject in vivo which method comprises detecting an ultrasound image of said target tissue wherein said target tissue is bound to a ligand specific for said target tissue and wherein said ligand is directly coupled to lipid-coated liquid perfluorocarbon particles contained in an emulsion, which method further comprises, prior to said detecting, administering said emulsion to said subject.
 2. The method of claim 1, wherein said lipid-coated liquid perfluorocarbon particles further comprise at least one chemotherapeutic agent or gene therapy delivery system.
 3. The method of claim 1, wherein said ligand comprises an antibody or fragment thereof, a virus, a receptor agonist or antagonist, a hormone, or a nucleic acid.
 4. The method of claim 3, wherein the ligand is an antibody or fragment thereof.
 5. A method for enhanced ultrasound imaging of a target tissue in a subject in vivo which method comprises administering to said subject an emulsion comprising a ligand specific for said target tissue, said ligand directly coupled to lipid-coated liquid perfluorocarbon particles, and detecting an ultrasound image of said target tissue wherein said target tissue is bound to said ligand and wherein said ligand is directly coupled to lipid-coated liquid perfluorocarbon particles contained in said emulsion.
 6. The method of claim 5, wherein said lipid-coated liquid perfluorocarbon particles further comprise at least one chemotherapeutic agent or gene therapy delivery system.
 7. The method of claim 5, wherein said ligand comprises an antibody or fragment thereof, a virus, a receptor agonist or antagonist, a hormone, or a nucleic acid.
 8. The method of claim 7, wherein the ligand is an antibody or fragment thereof.
 9. A method for enhanced ultrasound imaging of a target tissue in a subject in vivo which method comprises detecting an ultrasound image of said target tissue wherein said target tissue is bound to a ligand specific for said target tissue and wherein said ligand is directly coupled to lipid-coated liquid perfluorocarbon particles contained in an emulsion, wherein said subject has been administered said emulsion prior to said detecting.
 10. The method of claim 9, wherein said lipid-coated liquid perfluorocarbon particles further comprise at least one chemotherapeutic agent or gene therapy delivery system.
 11. The method of claim 9, wherein said ligand comprises an antibody or fragment thereof, a virus, a receptor agonist or antagonist, a hormone, or a nucleic acid.
 12. The method of claim 9, wherein the ligand is an antibody or fragment thereof. 